Abstract

Non-Hodgkin’s lymphomas comprise a group of heterogeneous lymphoid malignancies that represent the fifth most common form of cancer in the United States. A hallmark of many types of B-cell lymphomas is the constitutive expression of oncogenes such as the transcription factors Bcl-6, STAT3 and c-Myc and the anti-apoptotic protein Bcl-2. Over expression of these genes causes uncontrolled proliferation and survival of malignant cells, making knockdown of these genes by RNA interference (RNAi) a rational strategy for therapeutic intervention. RNAi is a conserved endogenous mechanism in which small interfering RNAs (siRNAs) suppress target-specific gene expression by promoting mRNA degradation. We have designed potent Dicer-substrate siRNAs using different computer algorithms to predict accessible target sites in the mRNAs of B-cell lymphoma oncogene targets. The Dicer-substrate 27mers are designed asymmetrically, so that Dicer processing yields a predicted 21mer siRNA duplex for entry into RNA induced silencing complex (RISC). Dicer-substrate siRNAs show improved efficacy at lower concentrations compared with conventional 21mer siRNAs, suggesting the reduction of potential off-target effects. In addition, we have designed bifunctional siRNA duplexes that contain two fully target-complimentary antisense strands against two different target mRNAs, but that are only partially complementary to each other. In vitro cleavage assays indicate that our bifunctional siRNAs have sufficient complementarity to form stable duplexes and can be processed into smaller molecules by recombinant Dicer. When delivered to the Burkitt’s lymphoma cell line Raji by electroporation, the most effective siRNAs reduced target mRNA levels by ∼80% as determined by quantitative RT-PCR and immunoblot analysis. Silencing of transcription factors affected the expression of downstream target genes, indicating a relevant effect on growth on survival of lymphoma cells through oncogene down regulation by RNAi. One concern with RNAi-mediated therapy is the possible recognition of siRNA duplex by cell’s own response to double-stranded RNA (dsRNA) that could trigger an unwanted interferon response. To determine whether our Dicer-substrate siRNAs cause an interferon response, we monitored induction of the cellular dsRNA pathway by measuring gene expression of p56, OAS1 and interferon by quantitative RT-PCR after transfection of siRNAs in different cell lines. None of the analyzed siRNAs show a significant increase in the expression of interferon pathway related genes indicating that our selected siRNAs are powerful silencers of gene expression without inducing an interferon response. In future studies, these new identified siRNAs will be incorporated in nanoparticles or attached to antibodies / aptamers for cell-specific delivery to lymphoma cell lines to evaluate their potential alone or in combination with chemotherapeutic drugs in therapy for lymphoma.

Author notes

Disclosure:Ownership Interests: John Rossi has options in Calando Pharmaceuticals and Benitec Inc. Membership Information: John Rossi is member of the Board of Directors of Cytogenix, Inc. Houston, Texas.